A practical guide to isolation of fluorescent Pseudomonas antagonic to Phytophthora palmivora (Butler) in Theobroma cacao L.

Black pod rot, caused by Phytophthora palmivora (Butler) is one of the most important diseases affecting the Theobroma cacao L. crop. The objective of the present work is to describe a methodology for the isolation and identification of fluorescent Pseudomonas antagonists of P. palmivora. Methods for collecting samples and obtaining bacterial isolates from the rhizosphere of T. cacao are described. The selection of antagonistic isolates is first carried out in a dual culture assay against a reference strain of P. palmivora, previously isolated from the same ecosystem. The strains selected for exhibiting plathogen's growth inhibition are submitted to a foliar test to determine in vivo antagonistic activity by treating detached leaves with the bacterial isolates and the pathogen. The bioprotection effect of bacteria against P. palmivora is also evaluated in planta under controlled conditions, by pre-treating roots with bacterial suspension and further inoculating the pathogen onto the leaves. The identification of bacterial isolates is performed using polyphasic taxonomy including physiological, biochemical and molecular methods. The methodology described here is a practical guide for selecting Pseudomonas with biocontrol potential against P. palmivora in the T. cacao agroecosystem.